The long-term goal of this project is to understand how CD1d controls the immune regulatory function of Va14Ja 15 NKT cells. In this application we propose to test the central hypothesis that the natural Va14Ja15 NKT cell antigen is a glycosphingolipid whose assembly with CD1d in MHC class lI-enriched vesicles (CIIV/MIIC) requires specialized protein chaperones. The rationale for this hypothesis is that CD1d controls the immune functions of NKT cells by presenting a putative self lipid antigen(s) whose chemical structure remains unknown. Currently, of all the lipid antigens known, only the synthetic anti-tumor agent alpha-galactosylceramide (alphaGalCer) activates almost all Va14Ja15 NKT cells. Our current data suggest that CD1d assembles with phospholipids in the ER and exchanges these ligands for another cellular lipid in the CIIV/MIIC. Thence the antigen-loaded CD1d is presented to NKT cells. The mechanism of exchange between cellular phospholipids and antigenic lipids remains undefined. Therefore, the chemical nature of the self lipid antigen(s) presented by CD1d to Va14Ja15 NKT cells and the mechanism(s) by which antigen loading occurs in the CIIV/MIIC currently remain outstanding unanswered questions of major immunological import. Our strategy to test the central hypothesis of this project will be to: a) Define the chemical structure of a natural Va14Ja15 NKT cell antigen to test the hypothesis that a natural Va14Ja15 NKT cell antigen is a glycosphingolipid. b) Define the chemical basis and immunological significance of Va14Ja15 NKT cell-antigen interaction and thereby test the hypothesis that alpha-glucosylceramide is a putative natural antigen. c) Determine the mechanism(s) underlying the assembly of CD1d with antigen to test the hypothesis that this assembly requires specialized protein chaperone(s). d) Define the molecular/structural features of the Va14Ja15 NKT cell receptor-antigen interface to test the hypothesis that TCR-lipid antigen interface bas unique features distinct that permit efficient antigen recognition at low concentrations. Thus the goals of this proposal have the potential of providing fundamental new insights into our understanding of immune recognition. In keeping with our long-term goal, upon completion of this project we expect to elucidate the mechanism by which CD1d1 assembles with antigen and controls Va14Ja15 NKT cell function. Additionally, the identification of a Va14Ja15 NKT cell antigen could lead to the development of natural therapeutics in place of the synthetic antigen alphaGalCer, which has been demonstrated to prevent the onset of several autoimmune diseases including type I diabetes and experimental autoimmune encephalomylitis. Together, they will significantly advance our understanding of the physiological role of the CD1 antigen presentation system and NKT cells within the context of the immune system.